Fauna and seasonality of sand flies (Diptera: Psychodidae: Phlebotominae) from a leishmaniasis transmission area in the central region of Rio Grande do Sul, Brazil

Abstract Sand flies, vectors capable of transmitting Leishmania spp. and causing leishmaniasis, have been a concern in the central region of Rio Grande do Sul, where canine leishmaniasis (CanL) has been documented since 1985. Notably, there has been a surge in CanL cases since 2017, with two autochthonous cases of human visceral leishmaniasis reported in the area in 2021. This study aimed to identify the sand fly fauna potentially involved in disease transmission. Modified Centers for Disease Control light traps were deployed in three neighborhoods of the city where CanL cases had been previously reported, spanning January 2021 to December 2022. Of the 89 collections conducted, 119 sand flies belonging to five species were captured: Pintomyia fischeri (76/119, 63.86%), Migonemyia migonei (23/119, 19.33%), Lutzomyia longipalpis (16/119, 13.45%), Brumptomyia sp. (2/119, 1.68%), and Psathyromyia lanei (2/119, 1.68%), predominantly between February and April in 2021 and 2022. Polymerase chain reaction testing on all female specimens yielded negative results for Leishmania spp. DNA. Although Leishmania spp. was not detected in these vectors, these findings underscore the imperative to implement measures aimed at curtailing the proliferation of these insects.

In urban areas, dogs (Canis familiaris) are the main infection reservoirs and sources.In the wild, foxes (Dusicyon vetulus and Cerdocyon thous) and marsupials (Didelphis albiventris and Didelphis marsupialis) also serve as reservoirs (Brasil, 2014).Studies have shown bat involvement in the protozoan lifecycle (Ratzlaff et al., 2022).In the state of RS, the first autochthonous cases occurred in dogs in 2008 and in humans the following year, with L. longipalpis identified as the main vector (Brasil, 2014).
According to the State Department of Health of Rio Grande do Sul, eight municipalities registered the presence of L. longipalpis until 2017.However, the municipalities of Viamão, Porto Alegre, and Santa Cruz do Sul did not register the main vector, with cases occurring in dogs and humans.The affected individuals lived close to forest fragments, and transmission was attributed to sand flies belonging to wild fauna (Rio Grande do Sul, 2017).In Porto Alegre, 777 CanL cases were reported between 2010and 2021, and 20 HVL cases were confirmed between 2016and 2021(Rio Grande do Sul, 2022).
Sporadic CanL cases have been reported in Santa Maria since 1985 (Pocai et al., 1998).However, as of 2017, there has been a considerable increase in CanL incidence (116 cases) and in 2021, two autochthonous human cases were recorded, one of which contributed to the patient's death (Wille, 2021).
According to the VL and CL Control Programs, recommended by the Ministry of Health, the objective of entomological investigations is to collect quantitative and qualitative information on transmitting sand flies in order to obtain new knowledge of the bioecology of insect species pertinent to human health (Brasil, 2014).Therefore, this study aimed to identify sand fly fauna potentially involved in the transmission of leishmaniasis, in a disease transmission area in the interior of the state of RS, and to present data related to the seasonal behavior of the main species found.

Materials and Methods
The municipality of Santa Maria (Figure 1) is located in central Rio Grande do Sul, southern Brazil, at 29°41'02"S and 53°48'25"W and an altitude of 113 m.The city has an area of 1,780.194km 2 and a population of 285,159 inhabitants (IBGE, 2021).
To capture the insects, modified CDC traps (Horst™ model) were installed in locations with previous CanL notifications from the Environmental Surveillance of the Municipality of Santa Maria, as shown in Figure 1.These locations were visited to request authorization from residents to install traps.Thus, three different locations were selected: Nossa Senhora do Perpétuo Socorro in the North (A), Presidente João Goulart in the Northeast (B), and Cerrito in the Center-East region (C) (Table 1).All sites were selected based on confirmation of dogs that presented anti-Leishmania spp.antibodies in rapid tests (TR-DPP®) and indirect immunofluorescence assay (IFA) (Varandas et al., 2001), as well as traps were installed according to the availability of residents to monitor and on days with no precipitation or strong winds.The traps were installed in the peridomicile, which were evaluated: the presence of domestic animals, organic matter, undergrowth, and fruit trees with shaded areas.The collections were carried out in the period between January 2021 and December 2022.The frequency of installing the traps was at least once a month.
The traps were placed approximately 1.5 m above the ground, activated at dusk (06:00 pm), and removed at dawn the next day (06:00 am), remaining uninterrupted for 12 h, coinciding with the feeding habits of sand flies.The devices were allocated at least once per month to each selected household.The temperature, precipitation, and humidity for the study period were obtained from the Instituto Nacional de Meteorologia (INMET, 2022).
After removing the traps, the collection bags were sent to the Parasitic Diseases Laboratory (LADOPAR) of the Universidade Federal de Santa Maria (UFSM) for captured insect selection.To dispatch the captured arthropods, the collection bag was stored in a freezer at -20 °C for 30 min.After this, all insects were examined under a stereoscopic microscope (Olympus®), and sand flies were screened according to external morphological characteristics, including size, presence of bristles throughout the body, and lanceolate wings (Galati, 2018).The remaining insects were discarded.
The selected specimens were sent to the Reservoir and Vector Laboratory of the Central Laboratory of the State of Rio Grande do Sul for morphological identification according to the taxonomic key proposed by Galati   2024; 33(3): e000824   4/9 Fauna and seasonality of Phlebotominae in Rio Grande do Sul, Brazil (2018).The results were tabulated in Excel spreadsheets, where sex, species, subspecies, locality of origin, climatic conditions, and environmental characteristics were recorded.
For molecular analysis, female flies were separated according to the date, place, and species found in each collection bag, then divided into individual samples or pools of up to 10 specimens.The samples were extracted using the Purelink® Genomic DNA Mini Kit (Invitrogen, USA) following the manufacturer's recommendations.The assessment of DNA quality and quantity extracted was conducted using the NanoDrop 1000 Spectrophotometer and measuring absorbance at 260/280 nm (Thermo Fisher Scientific).Subsequently, all samples were stored at -20 °C v PCR was performed.
The amplification reaction products were subjected to electrophoresis in 3% agarose gel (Ludwig Biotecnologia, Brazil), stained with SYBR safe DNA (Invitrogen, USA), stained with a UV transilluminator, and photographed for analysis.
To confirm inhibition of the reaction in PCR-negative samples, 0.25 μL DNA from a positive control sample was added to 0.25 μL DNA from a negative sample, and PCR was performed under the same conditions mentioned above.Additionally, the positive control were also diluted in the serial dilutions from 1:10 to 1:10 4 and retested, in order to determine the detection threshold.

Results
From January 2021 to December 2022, 89 collections were conducted: 32 in the Nossa Senhora do Perpétuo Socorro neighborhood (A), 28 in Presidente João Goulart (B), and 29 in Cerrito (C).The total population of sand flies captured in the municipality of Santa Maria, RS during the study period is shown in Table 2.In the Nossa Senhora do Perpétuo Socorro neighborhood (A), there was a stream with vegetation on the banks, abundant organic matter, humidity, and domestic cats; only L. longipalpis was found.The Presidente João Goulart neighborhood (B) collection site had an area shaded by bamboo (Bambusa taquara), organic matter, and domestic dogs, one of which was seropositive for Leishmania spp.during the study.Although this disease was not the primary cause of death in dogs, L. longipalpis and M. migonei were found.However, the Cerrito neighborhood (C) is adjacent to a vast, well-preserved residual Atlantic Forest, rich in organic matter and containing both domestic and wild animals, including wild opossums (Didelphis albiventris) and pheasants (Phasianus colchicus).In this locality, five species of sand flies were identified.
A graphic representation the seasonal distribution of sand flies collected in Santa Maria, RS, in relation to the average compensated temperature (°C) and total monthly precipitation between January 2021 and December 2022 is shown in Figure 2. The largest number (108/119, 90.75%) of specimens was collected in February, March, April, and December 2021, as well as February, March, April, and May 2022.These periods preceded or coincided with high rainfall and average temperatures > 20 °C.However, in periods with little rain and consequently low relative humidity or average temperatures < 20°C, the months with the lowest amount or no sand flies collected occurred.
Of the 81 female flies captured and divided into 30 individual samples or pools, all were tested using PCR and were negative for Leishmania spp.DNA.

Discussion
In the last 20 years, the southern region of Brazil has registered an increased number of CanL, and HVL cases (Rio Grande do Sul, 2022).Based on identification of the main vector (L.longipalpis), Souza et al. (2008) observed that protozoa were transmitted autochthonously in western Rio Grande do Sul.Furthermore, Rêgo et al. (2020) showed that in Porto Alegre, P. fischeri, M. migonei, and L. gaminarai may be associated with disease transmission in areas without L. longipalpis.
The main species transmitting VL in Brazil, L. longipalpis, was the third most abundant in this study (16/119, 13.44%), and the only one found in the three ecotopes studied.According to Brazil (2013), it is widely distributed in different ecological niches, especially in urban and rural areas.Because it is involved in the transmission of a disease of great social and economic relevance, and also because it is added to factors such as poor basic sanitation and accumulation of organic matter, it constitutes determining aspects for the maintenance of the vector in the environment.This data is very relevant, as L. longipalpis is considered the main vector of L. infantum, and although detected in smaller numbers in our study, it was found in all sampled points, being an important risk factor for the emergence of new cases in animals and humans.Therefore, entomological monitoring of potential L. infantum vectors is an important tool to prevent the spread of the disease.
Braz J Vet Parasitol 2024; 33(3): e000824 6/9 Fauna and seasonality of Phlebotominae in Rio Grande do Sul, Brazil In Santa Maria, VL cases have been reported since 1985, and two HVL cases were reported in 2021.However, until entomological studies were carried out, no sand fly species had been described in the region.In our study, all captured species were recorded in the state of RS; among these, M. migonei and P. fischeri are epidemiologically important because they have been implicated in L. braziliensis transmission, the main etiological agent of CL in Brazil (Shimabukuro & Galati, 2011).In several additional studies, they were implicated in infection with the etiological agent of VL, L. infantum (Guimarães et al., 2016).
In Santa Maria, P. fischeri (76/119, 63.86%) was the most frequently found species; however, it was only captured in the P3 ecotope, which is adjacent to a native forest environment.According to Rangel & Lainson (2003), P. fischeri has wild habitats, and its highest occurrence has been observed in areas of recent deforestation, especially where human habitation occurs.The species was naturally infected with Leishmania (Viannia) (Young & Duran, 1994) and L. braziliensis (Lana et al., 2015).Rêgo et al. (2020) reported the first molecular detection of L. infantum on P. fischeri in Porto Alegre and Galvis-Ovallos et al. ( 2021) confirmed its natural infection with L. infantum promastigotes in Embú das Artes, SP, focusing on CanL and HVL; this suggests it is a vector of this etiological agent, but does not confirm the vector potential (Alcover et al., 2012).
Migonemyia migonei was the second most common species (23/119, 19.32%) captured in the ecotope B (predominantly urbanized environment) and C (adjacent to native forest).This proves the ambience of this species, which was previously predominantly wild in urban environments.Guimarães et al. (2016) stated that this species is highly susceptible to L. infantum development, making it a permissive vector.According to Rangel & Lainson (2003), P. fischeri and M. migonei are notably anthropophilic, and according to Aguiar & Medeiros (2003), can also be captured in residual forests in marginal areas of cities, in annexes for domestic animals, and internal walls of human homes.
The main VL-transmitting species in Brazil, L. longipalpis, was the third most abundant in this study (16/119, 13.44%), and the only one found in all three surveyed ecotopes.According to Brazil (2013), it is widely distributed in several ecological niches, especially in urban and rural areas, where it has successfully established and proliferated, mainly due to anthropological environmental changes.This data is very relevant, as L. longipalpis is considered the main vector of L. infantum, and although detected in smaller numbers in our study, this vector was found at all sampled points.Therefore, entomological monitoring of potential vectors of L. infantum is crucial.
According to Aguiar & Medeiros (2003), Brumptomyia species are not important in leishmaniasis epidemiology.These sandflies are usually found in wild environments with leaves lying on the ground and reported in Dasipodidae (armadillo) burrows due to their food preferences.In our study, only two specimens of this genus were captured in the C ecotope.
Psathyromyia lanei (2/119, 1.68%) has wild and semi-domestic characteristics, inhabiting hollows and treetops in marginal areas and annexes of domestic animals.It is not associated with leishmaniasis transmission (Aguiar & Medeiros, 2003).This species was found only in C, which has all of these favorable conditions for its survival.Regarding insect seasonality, climatic factors, such as temperature, humidity, and rainfall, have a variable influence on sand fly populations, depending on the area studied (Dias et al., 2007).According to Souza et al. (2017), the temperature tends to be lower at the beginning of winter, causing a drop in the sand fly populations.However, our study also did not detect sanflies at higher temperatures, close to 25 °C (january 2021 and 2022).One potential explanation is "La Niña" phenomenon, which occurred both in 2021 and 2022 in RS, triggering long periods of drought, with rainfall below the historical average for the period.This may have negatively influenced the vector density, where the immature stages of these insects need organic matter and moisture to develop (Franke et al., 2002).Possibly, the intensity of rainfall as well as temperature variation may have influenced the entomological collection of vectors.PCR analysis indicated that the natural infection rate in sand flies may be low due to many factors, for example, resisting digestion by interfering with digestive enzymes (Telleria et al., 2010).In addition, Leishmania secrete a myoinhibitory peptide that interrupts hindgut peristalsis, delays fecal elimination, and increases the persistence of the parasite within the insect (Vaidyanathan, 2004).Leishmania cause damage to the stomodeal valve, interfering with the blood ingestion process, which can often lead to vector death (Volf et al., 2004).Although the PCR results of this study were negative, it is important to highlight that the study of these vectors is essential for understanding disease epidemiology in the region.
This study reinforces the evidence of autochthonous transmission of both VL and HVL and includes the municipality as a possible transmission area for CL, although such cases have not yet been reported.Based on this, Braz J Vet Parasitol 2024; 33(3): e000824 7/9 Fauna and seasonality of Phlebotominae in Rio Grande do Sul, Brazil we recommend carrying out entomological surveys covering more areas of the city and reinforce that preventive measures be adopted by public authorities, such as vector control, and by dog owners, such as using repellent collars.
This study verified that the sand fly fauna in the municipality of Santa Maria, central RS, is diverse, with the P. fischeri, M. migonei, L. longipalpis, Bumptomyia sp., and P. lanei being present.We found specimens of epidemiological interest that have not been previously described in the region, with a predominance of P. fischeri, M. migonei, and L. longipalpis.Based on these results and the detection of three sand fly species associated with VL and CL transmission, Santa Maria, RS represents an important focus for these diseases, both in dogs and humans.It is therefore important that sanitary measures are adopted, with the aim of providing information to the inhabitants of the region, as well as the implementation of public policies aimed at reducing insect proliferation, such as vector control and the implementation of basic sanitation.From this, other studies can be carried out in the municipality to identify the parasite and compare it with infections in humans and animals.In addition, measures may include health education in the affected neighborhoods, provision of repellent collars, as well as responsible ownership and custody of dogs and implementation of policies to prevent, control and combat the disease through the Sistema Único de Saúde.
of Phlebotominae in Rio Grande do Sul, Brazil

Figure 2 .
Figure 2. Seasonal distribution of sand flies (un), in relation to Average Compensated Temperature (°C) and Total Precipitation (mm), in the period between January 2021 and December 2022, in the municipality of Santa Maria, RS, Brazil.

Table 1 .
Description of sampling points and species of sand flies captured in the neighborhoods Nossa Senhora do Perpétuo Socorro, Presidente João Goulart and Cerrito in Santa Maria, RS, Brazil.